In many, if not most plant species, the development of hybrid cultivars is highly desired because of their generally increased productivity due to heterosis: the superiority of performance of hybrid individuals compared with their parents (see e.g., Fehr, 1987, Principles of cultivar development, Vol. 1: Theory and Technique, MacMillan Publishing Co., New York; Allard, 1960, Principles of Plant Breeding, John Wiley and Sons, Inc.).
The development of hybrid cultivars of various plant species depends upon the capability of achieving essentially almost complete cross-pollination between parents. This is most simply achieved by rendering one of the parent lines male sterile (i.e. bringing them in a condition so that pollen is absent or nonfunctional) either manually, by removing the anthers, or genetically by using, in the one parent, cytoplasmic or nuclear genes that prevent anther and/or pollen development (for a review of the genetics of male sterility in plants see Kaul, 1988, `Male Sterility in Higher Plants`, Springer Verlag).
For hybrid plants where the seed is the harvested product (e.g. corn, oilseed rape) it is in most cases also necessary to ensure that fertility of the hybrid plants is fully restored. In systems in which the male sterility is under genetic control this requires the existence and use of genes that can restore male fertility. The development of hybrid cultivars is mainly dependent on the availability of suitable and effective sterility and restorer genes.
Endogenous nuclear loci are known for most plant species that may contain genotypes which effect male sterility, and generally, such loci need to be homozygous for particular recessive alleles in order to result in a male-sterile phenotype. The presence of a dominant `male fertile` allele at such loci results in male fertility.
Recently it has been shown that male sterility can be induced in a plant by providing the genome of the plant with a chimeric male-sterility gene comprising a DNA sequence (or male-sterility DNA) coding, for example, for a cytotoxic product (such as an RNase) and under the control of a promoter which is predominantly active in selected tissue of the male reproductive organs. In this regard stamen-specific promoters, such as the promoter of the TA29 gene of Nicotiana tabacum, have been shown to be particularly useful for this purpose (Mariani et al., 1990, Nature 347:737, European patent publication ("EP") 0,344,029). By providing the nuclear genome of the plant with such a male-sterility gene, an artificial male-sterility locus is created containing the artificial male-sterility genotype that results in a male-sterile plant.
In addition it has been shown that male fertility can be restored to the plant with a chimeric fertility-restorer gene comprising another DNA sequence (or fertility-restorer DNA) that codes, for example, for a protein that inhibits the activity of the cytotoxic product or otherwise prevents the cytotoxic product from being active in the plant cells (European patent publication "EP" 0,412,911). For example the barnase gene of Bacillus amyloliquefaciens codes for an RNase, called barnase, which can be inhibited by a protein, barstar, that is encoded by the barstar gene of B. amylolicruefaciens. The barnase gene can be used for the construction of a sterility gene while the barstar gene can be used for the construction of a fertility-restorer gene. Experiments in different plant species, e.g. oilseed rape, have shown that a chimeric barstar gene can fully restore the male fertility of male sterile lines in which the male sterility was due to the presence of a chimeric barnase gene (EP 0,412,911, Mariani et al., 1991, Proceedings of the CCIRC Rapeseed Congress, Jul. 9-11, 1991, Saskatoon, Saskatchewan, Canada; Mariani et al., 1992, Nature 357:384). By coupling a marker gene, such as a dominant herbicide resistance gene (for example the bar gene coding for phosphinothricin acetyl transferase (PAT) that converts the herbicidal phosphinothricin to a non-toxic compound De Block et al., 1987, EMBO J. 6:2513!), to the chimeric male-sterility and/or fertility-restorer gene, breeding systems can be implemented to select for uniform populations of male sterile plants (EP 0,344,029; EP 0,412,911).
The production of hybrid seed of any particular cultivar of a plant species requires the: 1) maintenance of small quantities of pure seed of each inbred parent, and 2) the preparation of larger quantities of seed of each inbred parent. Such larger quantities of seed would normally be obtained by several (usually two) seed multiplication rounds, starting from a small quantity of pure seed ("basic seed") and leading, in each multiplication round, to a larger quantity of pure seed of the inbred parent and then finally to a stock of seed of the inbred parent (the "parent seed" or "foundation seed") which is of sufficient quantity to be planted to produce the desired quantities of hybrid seed. Of course, in each seed multiplication round larger planting areas (fields) are required.
In order to maintain and enlarge a small stock of seeds that can give rise to male-sterile plants it is necessary to cross the male sterile plants with normal pollen-producing parent plants. In the case in which the male-sterility is encoded in the nuclear genome, the offspring of such cross will in all cases be a mixture of male-sterile and male-fertile plants and the latter have to be removed from the former. With male-sterile plants containing an artificial male-sterility locus as described above, such removal can be facilitated by genetically linking the chimeric male sterility gene to a suitable marker gene, such as the bar gene, which allows the easy identification and removal of male-fertile plants (e.g. by spraying of an appropriate herbicide).
However, even when suitable marker genes are linked to male-sterility genotypes, the maintenance of parent male-sterile plants still requires at each generation the removal from the field of a substantial number of plants. For instance in systems using a herbicide resistance gene (e.g. the bar gene) linked to a chimeric male-sterility gene, as outlined above, only half of the parent stock will result in male-sterile plants, thus requiring the removal of the male-fertile plants by herbicide spraying prior to flowering. In any given field, the removal of male-fertile plants effectively reduces the potential yield of hybrid seed or the potential yield of male-sterile plants during each round of seed multiplication for producing parent seed. In addition removal of the male-fertile plants may lead to irregular stands of the male-sterile plants. For these reasons removal of the male-fertile plants is economically unattractive for many important crop species such as corn and oilseed rape.
Anthocyanins are pigments that are responsible for many of the red and blue colors in plants. The genetic basis of anthocyanin biosynthesis has been well characterized, particularly in corn, Petunia, and Antirrhinium (Dooner et al, 1991, Ann.Rev.Genet. 25:179-199; Jayaram and Peterson, 1990, Plant Breeding Reviews 2:91-137; Coe, 1994, In `The Maize Handbook`, Freeling and Walbot, eds. Springer Verlag New York Inc., p. 279-281). In corn anthocyanin biosynthesis is apparently under control of 20 or more genes. The structural loci C2, Whp, A1, A2, Bz1, and Bz2 code for various enzymes involved in anthocyanin biosynthesis and at least 6 regulatory loci, acting upon the structural genes, have been identified in corn i.e. the R, B, C1, Pl, P and Vp1 loci.
The R locus has turned out to be a gene family (in corn located on chromosome 10) comprising at least three different genes i.e. R (which itself may comprise duplicate genes organized in a tandem array), and the displaced duplicate genes R(Sn) and R(Lc). R typically conditions pigmentation of the aleurone but various alleles are known to confer distinct patterns of pigmentation. R(Lc) is associated with unique pigmentation of leaves and R(Sn) with unique pigmentation of the scutellar node. One state of R is associated with pigmentation of the whole plant (R(P)), while another is associated with pigmentation of the seeds (R(S)).
Alleles of the unlinked B locus (in corn located on chromosome 2) rarely condition pigmentation of the aleurone, but are frequently associated with pigmentation of mature plant parts. The B-peru allele however, pigments the aleurone (like R(S)). Analysis at the molecular level has confirmed that the R and B loci are duplicate genes.
In order that the R and B loci can color a particular tissue, the appropriate allele of C1 or Pl loci also needs to be present. The C1 and C1-S alleles, for instance, pigment the aleurone when combined with the suitable R or B allele.
Alleles of the C1 locus have been cloned and sequenced. of particular interest are C1 (Paz-Ares et al, 1987, EMBO J. 6:3553-3558) and C1-S (Schleffer et al, 1994, Mol.Gen.Genet. 242:40-48). Analysis of the sequences revealed the presence of two introns in the coding region of the gene. The protein encoded by the C1 and C1-S alleles shares homology with mvb proto-oncogenes and is known to be a nuclear protein with DNA-binding capacity acting as transcriptional activators.
The cDNA of the B-peru allele has also been analyzed and sequenced (Radicella et al, 1991, Plant Mol. Biol. 17:127-130). Genomic sequences of B-peru were also isolated and characterized based on the homology between R and B (Chandler et al., 1989, the Plant Cell 1:1175-1183; Radicella et al., 1992, Genes & Development 6:2152-2164). The tissue specificity of anthocyanin production of two different B alleles was shown to be due to differences in the promoter and untranslated leader sequences (Radicella et al, 1992, supra).
Various alleles of the R gene family have also been characterized at the molecular level, e.g. Lc (Ludwig et al, 1989, PNAS 86:7092-7096), R-nj, responsible for pigmentation of the crown of the kernel (Dellaporta et al, 1988, In "Chromosome Structure and Function,: Impact of New Concepts, 18th Stadeler Genetics Symposium, Gustafson and Appels, eds. (New York, Plenum press, pp. 263-282)), Sn (Consonni ei al, 1992, Nucl. Acids. Res. 20:373), and R(S) (Perrot and Cone, 1989, Nucl. Acids. Res. 17:8003).
The proteins encoded by the B and R genes share homology with mVc proto-oncogenes and have characteristics of transcriptional activators.
It has been shown that various structural and regulatory genes introduced in maize tissues by microprojectiles operate in a manner similar to the endogenous loci and can complement genotypes which are deficient in the introduced genes (Klein et al., 1989, PNAS 86:6681-6685; Goff et al., 1990, EMBO J. 9:2517-2522). The Lc gene was also used as a visible marker for plant transformation (Ludwig et al., 1990, Science 247:449-450). Apart from the above other genes involved in anthocyanin biosynthesis have been cloned (Cone, 1994, In `The Maize Handbook`, Freeling and Walbot eds., Springer Verlag New York Inc., p. 282-285).
In Barley, Falk et al (1981, In Barley Genetics IV, proceedings of the 4th International Barley Genetics symposium, Edinburgh University press, Edinburgh, pp. 778-785) have reported the coupling of a male-sterile gene to a xenia-expressing shrunken endosperm gene which makes it possible to select seeds, before planting, that will produce male-sterile plants. Problems associated with such proposal include complete linkage of the two genes (Stoskopf, 1993, Plant Breeding : Theory and Practice, Westview Press, Boulder, San Francisco, Oxford). In sweetcorn, a genetic system to produce hybrid corn seeds without detassling, which utilizes the closely linked genes y (white endosperm) and ms (male sterility) was suggested but was never used because of contamination from 5% recombination. Galinat (1975, J. Hered. 66:387-388) described a two-step seed production scheme that resolved this problem by using electronic color sorters to separate yellow from white kernels. This approach has not been utilized commercially (Kankis and Davis, 1986, in &lt;&lt;Breeding Vegetable Crops&gt;&gt;, the Avi Publishing Company Inc. Westport, Conn., U.S.A., p. 498).
EP 0,198,288 and U.S. Pat. No. 4,717,219 describe methods for linking marker genes (which can be visible markers or dominant conditional markers) to endogenous nuclear loci containing nuclear male-sterility genotypes.
EP 412,911 describes foreign restorer genes (e.g. barstar coding region under control of a stamen-specific promoter) that are linked to marker genes, including herbicide resistance genes and genes coding for pigments (e.g. the A1 gene) under control of a promoter which directs expression in specific cells, such as petal cells, leaf cells or seed cells, preferably in the outer layer of the seed.